Decoding links between the RNA polymerase II C-terminal domain and histone modifications

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The RNA polymerase II (RNAPII) elongation complex is a key hub for regulatory pathways controlling gene expression in eukaryotes. Aside from its intrinsic mRNA synthesis function, the RNAPII elongation complex governs co-transcriptional mRNA processing and export, covalent histone modifications, and chromatin structure. The molecular platform coordinating all of these functions is the C-terminal domain (CTD) of the RNAPII large subunit. This eukaryote-specific domain consists of multiple repeats of a conserved heptad motif (Y1S2P3T4S5P6S7). It is widely presumed that patterns of phosphorylation of CTD repeat residues within a given RNAPII constitute a “code” which dictates the constellation of factors with which it is engaged. Given the complex nature of the CTD, how factors directly recognize the CTD is, for the most part, poorly understood. This proposal aims to decipher the basis for CTD recognition by histone-modifying enzymes. Elongating RNAPII generates a highly stereotyped pattern of histone modifications at transcribed genes that correlates with specific phosphorylated forms of the CTD. How the CTD and chromatin modification patterns are coordinated in vivo, as well as the nature of the interactions between the RNAPII elongation complex and the relevant enzymes, are important unresolved issues. We will address these questions through the following specific aims: 1) Definition of the CTD “code” for select chromatin-modifying complexes using a library of synthetic peptides and a set of CTD mutants in the model eukaryote S. pombe. This will allow comprehensive assessment of the pattern of phosphorylations that regulate association of purified native complexes with CTD repeats. We will also carry out biochemical and structural characterization of complexes between chromatin modifiers and phosphorylated CTD peptides. 2) Investigation of the implementation and function of a CTD modification pathway regulating histone methylation in transcribed genes. In addition to deciphering the links between transcription and chromatin modification, we expect that our approach will yield general insights into how factors engage the RNAPII elongation complex.